Gibson Assembly or Isothermal Assembly Reaction

Isothermal cloning, more commonly known as Gibson assembly, efficiently joins multiple overlapping DNA fragments in a single-tube. It takes advantage of the interplay of 3 enzymes:

The 5’-exonuclease creates single-stranded 3´ overhangs into DNA fragments that facilitate the annealing of fragments that share complementarity at one end (overlap region).

The polymerase fills in gaps within each overlap region.

The DNA ligase seals nicks in the assembled DNA.

The final is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation. The downside of Gibson Assembly is that it does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.